Duplass has written, directed, and produced several feature films with his brother Jay Duplass. In 2005, he wrote and produced The Puffy Chair with his brother, in addition to portraying one of the main characters.

The two brothers later wrote, directed, and produced the films Baghead (2008) and Cyrus (2010) together[10] and have a unique style which consists of a great deal of ad-libbing off of the original script, shooting a number of takes, and editing scenes down 15 to 20 times.[11] Their films Jeff, Who Lives at Home and The Do-Deca-Pentathlon were released in 2012.[12][13] In 2014, Mark co-wrote, produced and starred in the horror filmCreep alongside Patrick Brice, Mark played the role of Josef. [14] In May 2016, it was announced that there would be a sequel to Creep. [15]Creep 2 was released in October 2017 which Mark co-wrote and starred in.

Mark starred in the FX comedy television series The League, with his wife, Katie Aselton. In 2015, the HBO series Togetherness debuted, which was created by and stars Duplass. That same year, Duplass co-starred with Evan Peters and Olivia Wilde in David Gelb's thriller film The Lazarus Effect.[16]

In 2018, Duplass was in a Wealthsimple commercial, he appeared in Tully and Duck Butter, and released his debut book Like Brothers alongside Jay Duplass.[17] He also starred as real estate developer Tom Wyatt in Season 2 of the Amazon Video series Goliath.